Antibody profiling sensitivity through increased reporter antibody layering

ABSTRACT

A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject&#39;s identity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. patentapplication Ser. No. 11/101,254, filed Apr. 6, 2005, which is adivisional of U.S. patent application Ser. No. 10/017,577, filed Dec.14, 2001, now U.S. Pat. No. 6,989,276, issued Jan. 24, 2006, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/290,256, filed May 10, 2001, the entire disclosure of each of whichis incorporated by reference herein.

CONTRACTUAL ORIGIN OF THE INVENTION

This invention was made with government support under Contract No.DE-AC07-94ID13223, Contract No. DE-AC07-99ID13727, and Contract No.DE-AC07-05ID14517 awarded by the United States Department of Energy. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to assaying biological samples. Moreparticularly, the invention relates to methods for analyzing biologicalsamples comprising antibody profiling. In an embodiment of theinvention, the analyzing of biological samples comprises a combinationof antibody profiling for characterizing individual-specific antibodiesin the biological samples and simultaneous assay of an analyte in thebiological samples.

BACKGROUND

Many methods are known for identifying individual or biological samplesobtained from such individuals. For example, blood typing is based onthe existence of antigens on the surface of red blood cells. The ABOsystem relates to four different conditions with respect to twoantigens, A and B. Type A individuals exhibit the A antigen; Type Bindividuals exhibit the B antigen; Type AB individuals exhibit both theA and B antigens; and Type O individuals exhibit neither the A nor the Bantigen. By analyzing a sample of a person's blood, it is possible toclassify the blood as belonging to one of these blood groups. While thismethod may be used to identify one individual out of a small group ofindividuals, the method is limited when the group of individuals islarger because no distinction is made between persons of the same bloodgroup. For example, the distribution of the ABO blood groups in the U.S.is approximately 45% O, 42% A, 10% B, and 3% AB. Tests based on otherblood group antigens or isozymes present in body fluids suffer from thesame disadvantages as the ABO blood typing tests. These methods canexclude certain individuals, but cannot differentiate between members ofthe same blood group.

A variety of immunological and biochemical tests based on genetics areroutinely used in paternity testing, as well as for determining thecompatibility of donors and recipients involved in transplant ortransfusion procedures, and also sometimes as an aid in theidentification of humans and animals. For example, serological testingof proteins encoded by the human leukocyte antigen (HLA) gene locus iswell known. Although a good deal of information is known concerning thegenetic makeup of the HLA locus, there are many drawbacks to using HLAserological typing for identifying individuals in a large group. Each ofthe HLA antigens must be tested for in a separate assay, and many suchantigens must be assayed to identify an individual, an arduous processwhen identifying one individual in a large group.

In the past decade, DNA-based analysis techniques, such as restrictionfragment length polymorphisms (RFLPs) and polymerase chain reaction(PCR) have rapidly gained acceptance in forensic and paternity analysesfor matching biological samples to an individual. RFLP techniques areproblematic, however, due to the need for relatively large sample sizes,specialized equipment, highly skilled technicians, and lengthy analysistimes. For forensic applications, there is often not enough sampleavailable for this type of assay, and in remote areas the necessaryequipment is often not available. In addition, this technique can takefrom two to six weeks for completion and can result in costly delays ina criminal investigation. Moreover, the cost of RFLP analysis can beprohibitory if screening of many samples is necessary. PCR techniqueshave advantages over RFLP analysis of requiring much smaller samplesizes and permitting more rapid analysis, but they still requirespecialized equipment and skilled technicians, and they are alsoexpensive.

U.S. Pat. No. 4,880,750 and U.S. Pat. No. 5,270,167 disclose “antibodyprofiling,” or “AbP,” as a method that purportedly overcomes many of thedisadvantages associated with DNA analysis. Antibody profiling is basedon the discovery that every individual has a unique set of antibodiespresent in his or her bodily fluids. R. M. Bernstein et al., “CellularProtein and RNA Antigens in Autoimmune Disease,” 2 Mol. Biol. Med.105-120 (1984). These antibodies, termed “individual-specificantibodies” or “ISAs,” have been found in blood, serum, saliva, urine,semen, perspiration, tears, and body tissues. A. M. Francoeur, “AntibodyFingerprinting: A Novel Method for Identifying Individual People andAnimals,” 6 Bio/Technology 821-825 (1988). ISAs are not associated withdisease and are thought to be directed against cellular components ofthe body. Every person is born with an antibody profile that matches themother's antibody profile. T. F. Unger and A. Strauss,“Individual-Specific Antibody Profiles as a Means of Newborn InfantIdentification,” 15 J. Perinatology 152-155 (1995). The child's antibodyprofile gradually changes, however, until a stable unique pattern isobtained by about two years of age. It has been shown that evengenetically identical individuals have different antibody profiles. Anindividual's profile is apparently stable for life and is not affectedby short-term illnesses. A. M. Francoeur, supra. Few studies have beenconducted on individuals with long-term diseases. Preliminary results,however, indicate that, although a few extra bands may appear, theoverall pattern remains intact. This technique has been used in themedical field to track patient samples and avoid sample mix-ups. Inaddition, the technique has been used in hospitals in cases whereswitching of infants or abduction has been alleged. The method has anumber of advantages over DNA techniques, including low cost, rapidanalysis (two hours from the time the sample is obtained), andsimplicity (no special equipment or training is necessary). In addition,this method will potentially work on samples that contain no DNA.

WO 97/29206 discloses a method for identifying the source of abiological sample used for diagnostic testing by linking diagnostic testresults to an antibody profile of the biological sample. By generatingan antibody profile of each biological sample, the origin of thebiological sample is identified.

Many assays are now available that use the attachment of specificnucleic acid probes or other biological molecules to surfaces such asglass, silicon, polymethacrylate, polymeric filters, microspheres,resins, and the like. In a configuration where the surface is planar,these assays are sometimes referred to as “biochips.” Initially,biochips contained nucleic acid probes attached to glass or siliconsubstrates in microarrays. These DNA chips are made by microfabricationtechnologies initially developed for use in computer chip manufacturing.Leading DNA chip technologies include an in situ photochemical synthesisapproach, P. S. Fodor, 277 Science 393-395 (1997); U.S. Pat. No.5,445,934; an electrochemical positioning approach, U.S. Pat. No.5,605,662; depositing gene probes on the chip using a sprayer thatresembles an ink-jet printer; and the use of gels in a solution-basedprocess. Arrays of other types of molecules, such as peptides, have beenfabricated on biochips, e.g., U.S. Pat. No. 5,445,934.

While the known methods for using antibody profiling are generallysuitable for their limited purposes, they possess certain inherentdeficiencies that detract from their overall utility in analyzing,characterizing, and identifying biological samples. For example, theknown methods rely on fractionation of antigens by electrophoresis andthen transfer of the fractionated antigens to a membrane. Due todifferences in conditions from one fractionation procedure to another,there are lot-to-lot differences in the positions of the antigens on themembrane such that results obtained using membranes from one lot cannotbe compared with results obtained using membranes from another lot.Further, when colorimetric procedures are used for detecting immunecomplexes on the membrane, color determination can be subjective suchthat results may be interpreted differently by different observers.

In view of the foregoing, providing a method for analyzing biologicalsamples, wherein lot-to-lot differences in reagents and subjectivity donot affect interpretation of results, would be a significant advancementin the art. More particularly, it would be advantageous to provide amethod for analyzing biological samples by antibody profiling in abiochip format such that analysis would be amenable to automation.

BRIEF SUMMARY OF THE INVENTION

One embodiment of the invention comprises a method for analyzingbiological material including individual-specific antibodies,comprising: forming an array of multiple antigens by attaching themultiple antigens to the surface of a solid support in a preselectedpattern such that the respective locations of the multiple antigens areknown; obtaining a sample of the biological material and contacting thearray with the sample such that a portion of the individual-specificantibodies contained in the sample reacts with and binds to antigens inthe array to form immune complexes; washing the solid support containingthe immune complexes such that antibodies in the sample that do notreact with and bind to the antigens in the array are removed; anddetecting the immune complexes and determining the locations thereofsuch that an antibody profile is obtained. In one embodiment, detectingthe immune complexes may be performed by exposing the immune complexesto a first additional antibody that recognizes and binds to theindividual-specific antibodies. In a further embodiment, one moreadditional antibodies that recognize the first additional antibody oreach other may be used.

According to embodiments of the invention, the detecting of the immunecomplexes comprises treating the solid support having immune complexesattached thereto such that the presence of immune complexes at alocation is characterized by a color change as compared to the absenceof immune complexes at the location. In one embodiment, the process ofdetecting the immune complexes further comprises monitoring the solidsupport with solid state color detection circuitry for comparing thecolor patterns before and after contacting the array with the sample. Inanother embodiment, the process of detecting the immune complexesfurther comprises obtaining a color camera image before and aftercontacting the array with the sample and analyzing pixel informationobtained therefrom. In still another embodiment of the invention, thesolid support is a surface plasmon resonance chip and the detecting ofthe immune complexes further comprises scanning the surface plasmonresonance chip before and after contacting the array with the sample andcomparing data obtained therefrom. In yet another embodiment of theinvention, the detecting of immune complexes comprises obtaining animage using a charge-coupled device to detect the color changecomprising fluorescence emission.

In yet another embodiment of the invention, the method is used as a testfor the use of drugs. Still another embodiment of the inventioncomprises analysis of an antibody profile obtained from a forensicsample and comparison with an antibody profile obtained from a samplefrom a criminal suspect or a victim of crime.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows illustrative antibody profiles obtained from saliva samplesaccording to the procedure of Example 1.

FIG. 2 shows comparisons of paired saliva and blood antibody profilesfrom five individuals according to the procedure of Example 1.

FIG. 3 shows antibody profiles obtained from saliva samples from asingle individual after contamination with various adulterants accordingto the procedure of Example 1.

FIG. 4 shows illustrative results obtained from immunoassay of Cocaine(crystalline tropane alkaloid) in saliva samples according to theprocedure of Example 1.

FIG. 5 shows illustrative results obtained from immunoassay ofmethamphetamine in saliva samples according to the procedure of Example1.

FIG. 6 shows illustrative results of immunodetection of Cocaine(crystalline tropane alkaloid) on a PVDF membrane: strip 5, 0 μg/mlCocaine (crystalline tropane alkaloid); strip 6, 0.1 μg/ml Cocaine(crystalline tropane alkaloid); strip 7, 10 μg/ml Cocaine (crystallinetropane alkaloid); and strip 8, 1000 μg/ml Cocaine (crystalline tropanealkaloid).

FIG. 7 shows illustrative results of immunodetection of methamphetamineon a PVDF membrane: strip 1, 0 μg/ml methamphetamine; strip 2, 0.1 μg/mlmethamphetamine; strip 3, 10 μg/ml methamphetamine; strip 4, 1000 μl/mlmethamphetamine.

FIG. 8 shows antibody profiles from three different individuals: onestrip of each pair contains no drugs; and the other strip of each paircontains 1000 μg/ml of Cocaine (crystalline tropane alkaloid) and ofmethamphetamine.

FIG. 9 shows antibody profiles for different amounts of serum using atwo-stage antibody layering process. Strip A was exposed to 50microliters of serum; strip B was exposed to 10 microliters of serum;strip C was exposed to 5 microliters of serum; strip D was exposed to 3microliters of serum; strip E was exposed to 1 microliter of serum;strip F was exposed to 0.5 microliter of serum; strip G was exposed to0.1 microliter of serum; and strip H was exposed to 0 microliter ofserum.

FIG. 10 shows antibody profiles for different amounts of serum using athree-stage antibody layering process. Strip A was exposed to 50microliters of serum; strip B was exposed to 25 microliters of serum;strip C was exposed to 15 microliters of serum; strip D was exposed to7.5 microliters of serum; strip E was exposed to 10 microliters ofserum; strip F was exposed to 2.5 microliters of serum; strip G wasexposed to 1 microliter of serum; strip H was exposed to 0.5 microliterof serum; strip I was exposed to 0.1 microliter of serum; and strip Jwas exposed to 0 microliter of serum.

FIG. 11 shows side-by-side antibody profiles of 3 microliters of serumwhere strip A is developed with a three-stage antibody layering processand strip B is developed with a two-stage antibody layering process.

FIG. 12 shows densitometry data from strips A and B of FIG. 11. The topline is strip A and the lower line is strip B.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods for analyzing biological samples aredescribed in detail, it is to be understood that this invention is notlimited to the particular configurations, process acts, and materialsdisclosed herein as such configurations, process acts, and materials mayvary somewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a method for analyzing a biological sample from “an animal”includes reference to two or more of such animals, reference to “a solidsupport” includes reference to one or more of such solid supports, andreference to “an array” includes reference to two or more of sucharrays.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “solid support” means a generally or substantiallyplanar substrate onto which an array of antigens is disposed. A solidsupport can comprise any material or combination of materials suitablefor carrying the array. Materials used to construct these solid supportsneed to meet several requirements, such as (1) a presence of surfacegroups that can be easily derivatized, (2) inertness to reagents used inthe assay, (3) stability over time, and (4) compatibility withbiological samples. For example, suitable materials include glass,silicon, silicon dioxide (i.e., silica), plastics, polymers, hydrophilicinorganic supports, and ceramic materials. Illustrative plastics andpolymers include poly(tetrafluoroethylene), poly(vinylidene difluoride),polystyrene, polycarbonate, polymethacrylate, and combinations thereof.Illustrative hydrophilic inorganic supports include alumina, zirconia,titania, and nickel oxide. An example of a glass substrate would be amicroscope slide. Silicon wafers used to make computer chips have alsobeen used to make biochips. See, for example, U.S. Pat. No. 5,605,662.

As used herein, “array” means an arrangement of locations on the solidsupport. The locations will generally be arranged in two-dimensionalarrays, but other formats are possible. The number of locations canrange from several to at least hundreds of thousands. The array patternand spot density can vary. For example, using a commercially availableGMS 417 Arrayer from Genetic MicroSystems, Inc. (Woburn, Mass.), thespot size and density can be selected by the user. With spots of 150 μmdiameter and 300 μm center-to-center spacing, more than 1000 spots canbe placed in a square centimeter and more than 10,000 spots can beplaced on a standard microscope slide. With 200 μm center-to-centerspacing, these numbers increase to 2500 per square centimeter and morethan 25,000 per slide.

As used herein, “colorigenic” refers to a substrate that produces acolored product upon digestion with an appropriate enzyme. Such coloredproducts include fluorescent and luminescent products.

A first act in the present method is to prepare an array of antigens byattaching the antigens to the surface of the solid support in apreselected pattern such that the locations of antigens in the array areknown. As used herein, an antigen is a substance that is bound by anantibody. Antigens can include proteins, carbohydrates, nucleic acids,hormones, drugs, receptors, tumor markers, and the like, and mixturesthereof. An antigen can also be a group of antigens, such as aparticular fraction of proteins eluted from a size exclusionchromatography column. Still further, an antigen can also be identifiedas a designated clone from an expression library or a random epitopelibrary.

In one embodiment of the invention, antigens are isolated from HeLacells as generally described in A.M. Francoeur et al., 136 J. Immunol.1648 (1986). Briefly, HeLa cells are grown in standard medium understandard tissue culture conditions. Confluent HeLa cell cultures arethen rinsed, preferably with phosphate-buffered saline (PBS), lysed withdetergent, and centrifuged to remove insoluble cellular debris. Thesupernate contains approximately 10,000 immunologically distinctantigens suitable for generating an array.

There is no requirement that the antigens used to generate the array beknown. All that is required is that the source of the antigens beconsistent such that a reproducible array can be generated. For example,the HeLa cell supernate containing the antigens can be fractionated on asize exclusion column, electrophoretic gel, density gradient, or thelike, as is well known in the art. Fractions are collected, and eachfraction collected could represent a unique set of antigens for thepurpose of generating the array. Thus, even though the antigens areunknown, a reproducible array can be generated if the HeLa cell antigensare isolated and fractionated using the same method and conditions.

Other methods, such as preparation of random peptide libraries orepitope libraries are well known in the art and may be used toreproducibly produce antigens (e.g., J. K. Scott and G. P. Smith,“Searching for Peptide Ligands with an Epitope Library,” 249 Science 386(1990); J. J. Devlin et al., “Random Peptide Libraries: A Source ofSpecific Protein Binding “Molecules,” 249 Science 404-406 (1990); S. E.Cwirla et al., “Peptides on Phage: A Vast Library of Peptides forIdentifying Ligands,” 87 Proc. Nat'l Acad. Sci. USA 6378-6382 (1990); K.S. Lam et al., “A New Type of Synthetic Peptide Library for IdentifyingLigand-binding Activity,” 354 Nature 82-84 (1991); S. Cabilly,“Combinatorial Peptide Library Protocols” (Humana Press, 304 pp.129-154, 1997); and U.S. Pat. No. 5,885,780). Such libraries can beconstructed by ligating synthetic oligonucleotides into an appropriatefusion phage. Fusion phages are filamentous bacteriophage vectors inwhich foreign sequences are cloned into phage gene III and displayed aspart of the gene III protein (pIII) at one tip of the virion. Each phageencodes a single random sequence and expresses it as a fusion complexwith pIII, a minor coat protein present at about five molecules perphage. For example, in the fusion phage techniques of J. K. Scott and G.P. Smith, supra, a library was constructed of phage containing avariable cassette of six amino acid residues. The hexapeptide modulesfused to bacteriophage proteins provided a library for the screeningmethodology that can examine >10¹² phages (or about 10⁸-10¹⁰ differentclones) at one time, each with a test sequence on the virion surface.The library obtained was used to screen monoclonal antibodies specificfor particular hexapeptide sequences. The fusion phage system has alsobeen used by other groups, and libraries containing longer peptideinserts have been constructed. Fusion phage prepared according to thismethodology can be selected randomly or non-randomly for inclusion inthe array of antigens. The fusion phages selected for inclusion in thearray can be propagated by standard methods to result in what isvirtually an endless supply of the selected antigens.

Other methods for producing antigens are also known in the art. Forexample, expression libraries can be prepared by random cloning of DNAfragments or cDNA into an expression vector (e.g., R. A. Young and R. W.Davis, “Yeast RNA Polymerase II Genes: Isolation with Antibody Probes,”222 Science 778-782 (1983); G. M. Santangelo et al., “Cloning of OpenReading Frames and Promoters from the Saccharomyces cerevisiae Genome:Construction of Genomic Libraries of Random Small Fragments,” 46 Gene181-186 (1986)). Expression vectors that could be used for making suchlibraries are commercially available from a variety of sources. Forexample, random fragments of HeLa cell DNA or cDNA can be cloned into anexpression vector, and then clones expressing HeLa cell proteins can beselected. These clones can then be propagated by methods well known inthe art. The expressed proteins are then isolated or purified and can beused in the making of the array.

Alternatively, antigens can be synthesized using recombinant DNAtechnology well known in the art. Genes that code for many viral,bacterial, and mammalian proteins have been cloned, and thus largequantities of highly pure proteins can be synthesized quickly andinexpensively. For example, the genes that code for many eukaryotic andmammalian membrane-bound receptors, growth factors, cell adhesionmolecules, and regulatory proteins have been cloned and are useful asantigens. Many proteins produced by such recombinant techniques, such astransforming growth factor, acidic and basic fibroblast growth factors,interferon, insulin-like growth factor, and various interleukins fromdifferent species, are commercially available.

In most instances, the entire polypeptide need not be used as anantigen. For example, any size or portion of the polypeptide thatcontains at least one epitope, i.e., antigenic determinant or portion ofan antigen that specifically interacts with an antibody, will sufficefor use in the array.

The antigens, whether selected randomly or non-randomly, are disposed onthe solid support to result in the array. The pattern of the antigens onthe solid support should be reproducible. That is, the location andidentity of each antigen on the solid support should be known. Forexample, in a 10×10 array one skilled in the art might place antigens1-100 in locations 1-100, respectively, of the array.

The proteins may placed in arrays on the surface of the solid supportusing a pipetting device or a machine or device configured for placingliquid samples on a solid support, for example, using a commerciallyavailable microarrayer, such as those from Cartesian Technologies, Inc.(Irvine, Calif.); Gene Machines (San Carlos, Calif.); GeneticMicroSystems, Inc. (Woburn, Mass.); GenePack DNA (Cambridge, UK);Genetix Ltd. (Christchurch, Dorset, UK); and Packard Instrument Company(Meriden, Conn.).

Relevant methods to array a series of protein antigens onto a surfaceinclude non-contact drop-on-demand dispensing and inkjet technology.Commercially available instruments are available for both methods.Cartesian Technologies offers several nanoliter dispensing instrumentsthat can dispense liquid volumes from 20 mL up to 250 μL from 96-, 384-,1536-, 3456-, and 9600-well microtiter plates and place them preciselyon a surface with densities up to 400 spots/cm². The instruments willspot onto surfaces in a variety of patterns. As the name implies, inkjettechnology utilizes the same principles as those used in inkjetprinters. MicroFab Technologies, Inc. (Plano, Tex.), offers a ten-fluidprint head that can dispense picoliter quantities of liquids onto asurface in a variety of patterns. An illustrative pattern for thepresent application would be a simple array ranging from 10×10 up to100×100.

There are a number of methods that can be used to attach proteins orother antigens to the surface of a solid support. The simplest of theseis simple adsorption through hydrophobic, ionic, and van der Waalsforces. This method is not optimal, however, since the proteins tend todetach from the surface over time. One suitable attachment chemistryinvolves the use of bifunctional organosilanes (e.g., Thompson andMaragos, 44 J. Agric. Food Chem. 1041-1046 (1996)). One end of theorganosilane reacts with exposed —OH groups on the surface of the chipto form a silanol bond. The other end of the organosilane contains agroup that is reactive with various groups on the protein surface, suchas —NH₂ and —SH groups. This method of attaching proteins to the chipresults in the formation of a covalent linkage between the protein andthe chip. Other suitable methods that have been used for proteinattachment to surfaces include arylazide, nitrobenzyl, and diazirinephotochemistry methodologies. Exposure of the above chemicals to UVlight causes the formation of reactive groups that can react withproteins to form a covalent bond. The arylazide chemistry forms areactive nitrene group that can insert into C—H bonds, while thediazirine chemistry results in a reactive carbene group. The nitrobenzylchemistry is referred to as caging chemistry whereby the caging groupinactivates a reactive molecule. Exposure to UV light frees the moleculeand makes it available for reaction. Still other methods for attachingproteins to solid supports are well known in the art, (e.g., S. S. Wong,Chemistry of Protein Conjugation and Cross-Linking CRC Press, 340,1991).

Following attachment of the antigens on the solid support in theselected array, the solid support should be washed by rinsing with anappropriate liquid to remove unbound antigens. Appropriate liquids forwashing include phosphate buffered saline (PBS) and the like, i.e.,relatively low ionic strength, biocompatible salt solutions buffered ator near neutrality. Many of such appropriate wash liquids are known inthe art or can be devised by a person skilled in the art without undueexperimentation (e.g., N. E. Good and S. Izawa, “Hydrogen Ion Buffers,”24 Methods Enzymology 53-68 (1972)).

The solid support is then processed for blocking of nonspecific bindingof proteins and other molecules to the solid support. This blocking stepprevents the binding of antigens, antibodies, and the like to the solidsupport wherein such antigens, antibodies, or other molecules are notintended to bind. Blocking reduces the background that might swamp outthe signal, thus increasing the signal-to-noise ratio. The solid supportis blocked by incubating the solid support in a medium that containsinert molecules that bind to sites where nonspecific binding mightotherwise occur. Examples of suitable blockers include bovine serumalbumin, human albumin, gelatin, nonfat dry milk, polyvinyl alcohol,TWEEN® 20, and various commercial blockers, such as SEABLOCK™ (trademarkof EastCoast Bio, Inc., North Berwick, Me.) and SUPERBLOCK® (trademarkof Pierce Chemical Co., Rockford, Ill.) blocking buffers.

Following washing for removal of unbound antigens from the array andblocking, the solid support is contacted with a liquid sample to betested. The sample can be from any animal that generatesindividual-specific antibodies. For example, humans, dogs, cats, mice,horses, cows, and rabbits have all been shown to possess ISAs. Thesample can be from various bodily fluids and solids, including blood,saliva, semen, serum, plasma, urine, amniotic fluid, pleural fluid,cerebrospinal fluid, and mixtures thereof. These samples are obtainedaccording to methods well known in the art. Depending on the detectionmethod used, it may be required to manipulate the biological sample toattain optimal reaction conditions. For example, the ionic strength orhydrogen ion concentration or the concentration of the biological samplecan be adjusted for optimal immune complex formation, enzymaticcatalysis, and the like.

As described in detail in U.S. Pat. No. 5,270,167 to Francoeur, whenISAs are allowed to react with a set of random antigens, a certainnumber of immune complexes form. For example, using a panel of about1000 unique antigens, about 30 immune complexes between ISAs in abiological sample that has been diluted twenty-fold can be detected. Ifthe biological sample is undiluted, the total number of possibledetectable immune complexes that could form would be greater than 10²³.The total number of possible immune complexes can also be increased byselecting “larger” antigens, i.e., proteins instead of peptides) thathave multiple epitopes. Therefore, it will be appreciated that dependingon the antigens and number thereof used, the dilution of the biologicalsample, and the detection method, one skilled in the art can regulatethe number of immune complexes that will form and be detected. The setof unique immune complexes that form and fail to form between the ISAsin the biological sample and the antigens in the array constitute anantibody profile.

Methods for detecting antibody/antigen or immune complexes are wellknown in the art. The present invention can be modified by one skilledin the art to accommodate the various detection methods known in theart. The particular detection method chosen by one skilled in the artdepends on several factors, including the amount of biological sampleavailable, the type of biological sample, the stability of thebiological sample, the stability of the antigen, and the affinitybetween the antibody and antigen. Moreover, as discussed above,depending on the detection methods chosen, it may be required to modifythe biological sample.

While these techniques are well known in the art, examples of a few ofthe detection methods that may be used to practice the present inventionare briefly described below.

There are many types of immunoassays known in the art. The most commontypes of immunoassay are competitive and non-competitive heterogeneousassays, such as enzyme-linked immunosorbent assays (ELISAs). In anon-competitive ELISA, unlabeled antigen is bound to a solid support,such as the surface of the biochip. Biological sample is combined withantigens bound to the reaction vessel, and antibodies (primaryantibodies) in the biological sample are allowed to bind to theantigens, forming the immune complexes. After the immune complexes haveformed, excess biological sample is removed and the biochip is washed toremove nonspecifically bound antibodies. The immune complexes may thenbe reacted with an appropriate enzyme-labeled anti-immunoglobulin(secondary antibody). The secondary antibody reacts with antibodies inthe immune complexes, not with other antigens bound to the biochip.Secondary antibodies specific for binding antibodies of differentspecies, including humans, are well known in the art and arecommercially available, such as from Sigma Chemical Co. (St. Louis, Mo.)and Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). After a furtherwash, the enzyme substrate is added. The enzyme linked to the secondaryantibody catalyzes a reaction that converts the substrate into aproduct. When excess antigen is present, the amount of product isdirectly proportional to the amount of primary antibodies present in thebiological sample. The product may be fluorescent or luminescent, whichcan be measured using technology and equipment well known in the art. Itis also possible to use reaction schemes that result in a coloredproduct, which can be measured spectrophotometrically.

In other embodiments of the invention, the secondary antibody may not belabeled to facilitate detection. Additional antibodies may be layered(i.e., tertiary, quaternary, etc.) such that each additional antibodyspecifically recognizes the antibody previously added to the immunecomplex. Any one of these additional (i.e., tertiary, quaternary, etc.)may be labeled so as to allow detection of the immune complex asdescribed herein.

Sandwich or capture assays can also be used to identify and quantifyimmune complexes. Sandwich assays are a mirror image of non-competitiveELISAs in that antibodies are bound to the solid phase and antigen inthe biological sample is measured. These assays are particularly usefulin detecting antigens having multiple epitopes that are present at lowconcentrations. This technique requires excess antibody to be attachedto a solid phase, such as the biochip. The bound antibody is thenincubated with the biological samples, and the antigens in the sampleare allowed to form immune complexes with the bound antibody. The immunecomplex is incubated with an enzyme-linked secondary antibody, whichrecognizes the same or a different epitope on the antigen as the primaryantibody. Hence, enzyme activity is directly proportional to the amountof antigen in the biological sample. D. M. Kemeny and S. J. Challacombe,“ELISA and Other Solid Phase Immunoassays” (1988).

Typical enzymes that can be linked to secondary antibodies includehorseradish peroxidase, glucose oxidase, glucose-6-phosphatedehydrogenase, alkaline phosphatase, β-galactosidase, and urease.Secondary antigen-specific antibodies linked to various enzymes arecommercially available from, for example, Sigma Chemical Co. andAmersham Life Sciences (Arlington Heights, Ill.).

Competitive ELISAs are similar to noncompetitive ELISAs except thatenzyme linked antibodies compete with unlabeled antibodies in thebiological sample for limited antigen binding sites. Briefly, a limitednumber of antigens are bound to the solid support. Biological sample andenzyme-labeled antibodies are added to the solid support.Antigen-specific antibodies in the biological sample compete withenzyme-labeled antibodies for the limited number of antigens bound tothe solid support. After immune complexes have formed, nonspecificallybound antibodies are removed by washing, enzyme substrate is added, andthe enzyme activity is measured. No secondary antibody is required.Because the assay is competitive, enzyme activity is inverselyproportional to the amount of antibodies in the biological sample.

Another competitive ELISA can also be used within the scope of thepresent invention. In this embodiment, limited amounts of antibodiesfrom the biological sample are bound to the surface of the solid supportas described herein. Labeled and unlabeled antigens are then broughtinto contact with the solid support such that the labeled and unlabeledantigens compete with each other for binding to the antibodies on thesurface of the solid support. After immune complexes have formed,nonspecifically bound antigens are removed by washing. The immunecomplexes are detected by incubation with an enzyme-linked secondaryantibody, which recognizes the same or a different epitope on theantigen as the primary antibody, as described above. The activity of theenzyme is then assayed, which yields a signal that is inverselyproportional to the amount of antigen present.

Homogeneous immunoassays can also be used when practicing the method ofthe present invention. Homogeneous immunoassays may be preferred fordetection of low molecular weight compounds, such as hormones,therapeutic drugs, and illegal drugs that cannot be analyzed by othermethods, or compounds found in high concentration. Homogeneous assaysare particularly useful because no separation step is necessary. R. C.Boguslaski et al., “Clinical Immunochemistry: Principles of Methods andApplications” (1984).

In homogeneous techniques, bound or unbound antigens are enzyme-linked.When antibodies in the biological sample bind to the enzyme-linkedantigen, steric hindrances inactivate the enzyme. This results in ameasurable loss in enzyme activity. Free antigens (i.e., notenzyme-linked) compete with the enzyme-linked antigen for limitedantibody binding sites. Thus, enzyme activity is directly proportionalto the concentration of antigen in the biological sample.

Enzymes useful in homogeneous immunoassays include lysozyme,neuraminidase, trypsin, papain, bromelain, glucose-6-phosphatedehydrogenase, and β-galactosidase. T. Persoon, “Immunochemical Assaysin the Clinical Laboratory,” 5 Clinical Laboratory Science 31 (1992).Enzyme-linked antigens are commercially available or can be linked usingvarious chemicals well known in the art, including glutaraldehyde andmaleimide derivatives.

Prior antibody profiling technology involves an alkaline phosphataselabeled secondary antibody with 5-bromo-4-chloro-3′-indolylphosphatep-toluidine salt (BCIP) and nitro-blue tetrazolium chloride (NBT), bothof which are commercially available from a variety of sources, such asfrom Pierce Chemical Co. (Rockford, Ill.). The enzymatic reaction formsan insoluble colored product that is deposited on the surface of themembrane strips to form bands wherever antigen-antibody complexes occur.This method is suboptimal in a biochip format since it is difficult toquantify and since colorimetric methods are typically less sensitivethan assays based on fluorescence or luminescence.

Fluorescent immunoassays can also be used when practicing the method ofthe present invention. Fluorescent immunoassays are similar to ELISAsexcept the enzyme is substituted for fluorescent compounds calledfluorophores or fluorochromes. These compounds have the ability toabsorb energy from incident light and emit the energy as light of alonger wavelength and lower energy. Fluorescein and rhodamine, usuallyin the form of isothiocyanates that can be readily coupled to antigensand antibodies, are most commonly used in the art. D. P. Stites et al.,“Basic and Clinical Immunology” (1994). Fluorescein absorbs light of 490nm to 495 nm in wavelength and emits light at 520 nm in wavelength.Tetramethylrhodamine absorbs light of 550 nm in wavelength and emitslight at 580 nm in wavelength. Illustrative fluorescence-based detectionmethods include ELF-97 alkaline phosphatase substrate (Molecular Probes,Inc., Eugene, Oreg.); PBXL-1 and PBXL-3 (phycobilisomes conjugated tostreptavidin) (Martek Biosciences Corp., Columbia, Md.); FITC(fluorescein isothiocyanate) and Texas Red labeled goat anti-human IgG(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.); andB-Phycoerythrin and R-Phycoerythrin conjugated to streptavidin(Molecular Probes, Inc.). ELF-97 is a nonfluorescent chemical that isdigested by alkaline phosphatase to form a fluorescent molecule. Becauseof turnover of the alkaline phosphatase, use of the ELF-97 substrateresults in signal amplification. Fluorescent molecules attached tosecondary antibodies do not exhibit this amplification.

Phycobiliproteins isolated from algae, porphyrins, and chlorophylls,which all fluoresce at about 600 nm, are also being used in the art. I.Hemmila, “Fluoroimmunoassays and Immunofluorometric Assays,” 31 Clin.Chem. 359 (1985); U.S. Pat. No. 4,542,104. Phycobiliproteins andderivatives thereof are commercially available under the namesR-phycoerythrin (PE) and QUANTUM RED™ from, for example, Sigma ChemicalCo.

In addition, Cy-conjugated secondary antibodies and antigens are usefulin immunoassays and are commercially available. Cy3, for example, ismaximally excited at 554 nm and emits light at between 568 nm and 574nm. Cy3 is more hydrophilic than other fluorophores and thus has less ofa tendency to bind nonspecifically or aggregate. Cy-conjugated compoundsare commercially available from Amersham Life Sciences.

Illustrative luminescence-based detection methods include CSPD® and CDPstar alkaline phosphatase substrates (Roche Molecular Biochemicals,Indianapolis, Ind.); and SUPERSIGNAL® horseradish peroxidase substrate(Pierce Chemical Co., Rockford, Ill.).

Chemiluminescence, electroluminescence, and electrochemiluminescence(ECL) detection methods are also attractive means for quantifyingantigens and antibodies in a biological sample. Luminescent compoundshave the ability to absorb energy, which is released in the form ofvisible light upon excitation. In chemiluminescence, the excitationsource is a chemical reaction; in electroluminescence the excitationsource is an electric field; and in ECL an electric field induces aluminescent chemical reaction.

Molecules used with ECL detection methods generally comprise an organicligand and a transition metal. The organic ligand forms a chelate withone or more transition metal atoms forming an organometallic complex.Various organometallic and transition metal-organic ligand complexeshave been used as ECL labels for detecting and quantifying analytes inbiological samples. Due to their thermal, chemical, and photochemicalstability, their intense emissions and long emission lifetimes,ruthenium, osmium, rhenium, iridium, and rhodium transition metals arefavored in the art. The types of organic ligands are numerous andinclude anthracene and polypyridyl molecules and heterocyclic organiccompounds. For example, bipyridyl, bipyrazyl, terpyridyl, andphenanthrolyl, and derivatives thereof, are common organic ligands inthe art. A common organometallic complex used in the art includestris-bipyridine ruthenium (II), commercially available from IGEN, Inc.(Rockville, Md.) and Sigma Chemical Co.

Advantageously, ECL can be performed under aqueous conditions and underphysiological pH, thus minimizing biological sample handling. J. K.Leland et al., “Electrogenerated Chemiluminescence: AnOxidative-Reduction Type ECL Reactions Sequence Using Triprophyl Amine,”137 J. Electrochemical Soc. 3127-3131 (1990); WO 90/05296; and U.S. Pat.No. 5,541,113. Moreover, the luminescence of these compounds may beenhanced by the addition of various cofactors, such as amines.

In practice, a tris-bipyridine ruthenium (II) complex, for example, maybe attached to a secondary antibody using strategies well known in theart, including attachment to lysine amino groups, cysteine sulfhydrylgroups, and histidine imidazole groups. In a typical ELISA immunoassay,secondary antibodies would recognize ISAs bound to antigens, but notunbound antigens. After washing nonspecific binding complexes, thetris-bipyridine ruthenium (II) complex would be excited by chemical,photochemical, and electrochemical excitation means, such as by applyingcurrent to the biochip (e.g., WO 86/02734). The excitation would resultin a double oxidation reaction of the tris-bipyridine ruthenium (II)complex, resulting in luminescence that could be detected by, forexample, a photomultiplier tube. Instruments for detecting luminescenceare well known in the art and are commercially available, for example,from IGEN, Inc. (Rockville, Md.).

Solid state color detection circuitry can also be used to monitor thecolor reactions on the biochip and, on command, compare the colorpatterns before and after the sample application. A color camera imagecan also be used and the pixel information analyzed to obtain the sameinformation.

Still another method involves detection using a surface plasmonresonance (SPR) chip. The surface of the chip is scanned before andafter sample application and a comparison is made. The SPR chip relieson the refraction of light when the molecules of interest are exposed toa light source. Each molecule has its own refraction index by which itcan be identified. This method requires precise positioning and controlcircuitry to scan the chip accurately.

Yet another method involves a fluid rinse of the biochip with afluorescing reagent. The microlocations that combine with the biologicalsample will fluoresce and can be detected with a charge-coupled device(CCD) array. The output of such a CCD array is analyzed to determine theunique pattern associated with each sample. This approach avoids theproblems associated with scanning technologies. Speed is not a factorwith any of the methods since the chemical combining of sample andreference takes minutes to occur.

Moreover, array scanners are commercially available, such as fromGenetic MicroSystems, Inc. The GMS 418 Array Scanner uses laser opticsto rapidly move a focused beam of light over the biochip. This systemuses a dual-wavelength system including high-powered, solid-state lasersthat generate high excitation energy to allow for reduced excitationtime. At a scanning speed of 30 Hz, the GMS 418 can scan a 22×75-mmslide with 10-μm resolution in about four minutes.

Software for image analysis obtained with an array scanner is readilyavailable. Available software packages include IMAGENE® (BioDiscovery,Los Angeles, Calif.); ScanAlyze (available at no charge; developed byMike Eisen, Stanford University, Palo Alto, Calif.); De-Array (developedby Yidong Chen and Jeff Trent of the National Institutes of Health; usedwith IP Lab from Scanalytics, Inc., Fairfax, Va.); Pathways (ResearchGenetics, Huntsville, Ala.); GEM Tools (Incyte Pharmaceuticals, Inc.,Palo Alto, Calif.); and Imaging Research (Amersham Pharmacia Biotech,Inc., Piscataway, N.J.).

Once interactions between the antigens and ISAs have been identified andquantified, the signals may be digitized. The digitized antibody profileserves as a signature that identifies the source of the biologicalsample. Depending on the biochip used, the digitized data may takenumerous forms. For example, the biochip may comprise an array with 10columns and 10 rows for a total number of 100 microlocations. Eachmicrolocation contains at least one antigen. After the biological samplecontaining the ISAs is added to each microlocation and allowed toincubate, interactions between antigens and ISAs in the biologicalsample are identified and quantified. In each microlocation, aninteraction between the antigen at that microlocation and the ISAs inthe biological sample either do or do not result in a quantifiablesignal. In one embodiment, the results of the antibody profile aredigitized by ascribing each one of the 100 microlocations a numericalvalue of either “0,” if a quantifiable signal was not obtained, or “1,”if a quantifiable signal was obtained. Using this method, the digitizedantibody profile comprises a unique set of zeroes and ones.

The numerical values “0” or “1” may, of course, be normalized to signalsobtained in internal control microlocations so that digitized antibodyprofiles obtained at a later time can be properly compared. For example,one or several of the microlocations will contain a known antigen, whichwill remain constant over time. Therefore, if a subsequent biologicalsample is more or less dilute than a previous biological sample, thesignals can be normalized using the signals from the known antigen.

It will be appreciated by one skilled in the art that other methods ofdigitizing the antibody profile exist and may be used. For example,rather than ascribing each microlocation with a numerical value of “0”or “1,” the numerical value may be incremental and directly proportionalto the strength of the signal.

By digitizing the antibody profile signals, the biochemical results canbe entered into a computer and quickly accessed and referenced. Withinseconds of having the antibody profile digitized, a computer can comparea previously digitized antibody profile to determine whether there is amatch. If a matching antibody profile is in the database, a positiveidentification of the source of the biological sample can be made. Thus,the method of the present invention can both discriminate and positivelyidentify the source of a biological sample.

In an embodiment of the invention, the present method is used forforensic analysis for matching a biological sample to a criminalsuspect. Forensic samples obtained from crime scenes are often subjectto drying of the samples, small sample sizes, mixing with samples frommore than one individual, adulteration with chemicals, and the like. Thepresent method provides the advantages of rapid analysis, simplicity,low cost, and accuracy for matching forensic samples with suspects. Forexample, the forensic sample and a sample from one or more suspects areobtained according to methods well known in the art. Antibody profilesfor each of the samples are prepared, as described herein. The antibodyprofiles are then compared. A match of antibody profiles means that theforensic sample was obtained from the matching suspect. If no match ofantibody profiles is obtained, then none of the suspects was the sourceof the forensic sample.

In another embodiment of the invention, the present method is used fordrug testing of individuals. For example, in many work places it is acondition of obtaining or maintaining employment to be free of illegaldrug use. The presence of illegal drugs in the bloodstream of a personcan be detected by the present method by antibody capture or similarmethods. Moreover, as described in WO 97/29206, the drug test and theidentity of the sample can be correlated in a single test. Drug testsare also important in certain animals, such as horses and dogs involvedin racing.

The present invention is further described in the following examples,which are offered by way of illustration and are not limiting of theinvention in any manner.

EXAMPLE 1

The law enforcement community has demonstrated several needs associatedwith drug testing of suspects including dealing with privacy issuesassociated with sample collection, maintenance of sample chain ofcustody, prevention of sample adulteration by the suspect, andfacilitating more rapid turnaround time on sample analyses. Current drugtesting protocols utilize urine samples and, occasionally, bloodsamples. Invasion of privacy is a continuing problem with urine samplessince it is necessary to observe the individual providing the sample tomaintain the chain of custody and eliminate the possibility of sampleswitching or adulteration. Urine samples are also not a good indicatorof the current level of intoxication since many drug metabolitescontinue to be excreted into urine for days or weeks after the drugs areinitially taken. While blood samples do not suffer from these problems,collecting blood is an invasive procedure requiring special facilitiesand trained personnel that may not always be available when the needarises. It is necessary for law enforcement personnel to maintain strictchain of custody for all samples collected to ensure that mishandling ordeliberate tampering do not occur. A break or even a perceived break inthe chain of custody can result in evidence being dismissed outright orgiven little weight.

Embodiments of the present invention solve these issues in several ways.First, incorporation of the antibody profiling identification assay intothe drug test makes identification of the sample donor integral to thetest and eliminates the need for complex chain of custody procedures.Second, a saliva-based drug test is better than a urine test becausedrug levels in saliva can be readily correlated with drug levels inblood (W. Schramm et al., “Drugs of Abuse in Saliva: A Review,” 16 J.Analy. Toxicology 1-9 (1992); E. J. Cone, “Saliva Testing for Drugs ofAbuse,” 694 Ann. N.Y. Acad. Sci. 91-127 (1995)), therefore providing abetter indicator of current drug use (D. A. Kidwell et al., “Testing forDrugs of Abuse in Saliva and Sweat,” 713 J. of Chromatography B:Biomedical Sciences and Applications 111-135 (1998)). Saliva samplesfrom a suspect can also be collected easily in view of a law enforcementofficer without invasion of privacy or with invasive methods. Finally,the present test is easy to use and can be quickly performed by lawenforcement personnel on site, instead of requiring the days to weeksnecessary at distant centralized laboratories. V. S. Thompson et al.,“Antibody Profiling as an Identification Tool for Forensic Samples,”3576 Investigation and Forensic Science Technologies 52-59 (1999).

In this example, an antibody-based test is provided for two commonillicit drugs (Cocaine (crystalline tropane alkaloid) andmethamphetamine). These drugs are among the most commonly abused, andtheir use is on the rise. S. B. Karch, Drug Abuse Handbook (CRC Press,1998); L. D. Bowers, “Athletic Drug Testing, 17 Sports Pharmacology,”299-318 (1998).

Materials and Methods. Goat anti-rabbit IgG antibodies conjugated toalkaline phosphatase were obtained from Jackson ImmunoResearch (WestGrove, Pa.). Rabbit anti-human IgA antibodies were purchased from U.S.Biological (Swampscott, Mass.). SEABLOCK™, nitro-blue tetrazoliumchloride/5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt(NBT/BCIP), p-nitrophenyl phosphate disodium salt (PNPP), EZ-LINK®maleimide activated alkaline phosphatase kits, and FREEZYME™ conjugatepurification kits were obtained from Pierce Chemical (Rockford, Ill.).Monoclonal antibodies against benzoylecgonine and methamphetamine, andbovine serum albumin (BSA) conjugates of methamphetamine andbenzoylecgonine were purchased from O.E.M. Concepts (Toms River, N.J.).Cocaine (crystalline tropane alkaloid) and methamphetamine hydrochloridesalts were obtained from Sigma-Aldrich (St. Louis, Mo.). AntibodyProfiling strips were purchased from Miragen, Inc. (Irvine, Calif.).Strips used for the combined drug-AbP test were produced according tothe protocol of A. M. Francoeur, “Antibody Fingerprinting: A NovelMethod for Identifying Individual People and Animals,” 6 Bio/Technology822-825 (1988). Saliva samplers from Saliva Diagnostic Systems, Inc.(Vancouver, Wash.), OraSure Technologies, Inc. (Bethlehem, Pa.), andSarstedt, Inc. (Newton, N.C.), were used to collect saliva samples fromvolunteers.

A saliva-based AbP assay was developed through modification of anearlier protocol designed for processing blood samples. T. F. Unger andA. Strauss, “Individual-Specific Antibody Profiles as a Means of NewbornInfant Identification,” 15 J. Perinatology 152-155 (1995). Briefly, 500μl of saliva sample diluted with 1.0 ml of PBST (50 mM phosphatebuffered saline, 0.2% TWEEN® 20) was incubated with an AbP stripovernight for a minimum of 16 hours, and excess sample was washed offwith PBST. Next, the strip was incubated successively with 100 ng/mlrabbit anti-human IgA for 1 hour and 100 ng/ml goat anti-rabbitIgG-alkaline phosphatase conjugate for 30 minutes with washes in betweenincubations. The strip was washed again with PBST and a precipitationsubstrate for alkaline phosphatase, NBT/BCIP, was added to allowdevelopment of bands on the strip.

The SALIVA SAMPLER™ (Saliva Diagnostic Systems) and the SALIVETTE™(Sarstedt, Inc.) saliva collection systems were examined forcompatibility with the AbP assay. The SALIVA SAMPLER™ system comprises acotton pad attached to a plastic handle. A window in the handle turnsblue when sufficient sample has been collected. The pad is placed in apreservative buffer after collection. The SALIVETTE™ is a cotton rollplaced in the mouth for about 10 minutes and then centrifuged in aplastic tube to collect a sample. Both types of samplers were placed inthe gingival crevice of the mouth for sample collection. The quality ofsamples as a function of storage time at temperatures of −20° C., 4° C.,and 25° C. was assessed by performing AbP on samples collected with bothsamplers.

Five volunteers participated in studies to compare blood AbP patternswith those obtained from saliva samples. Protocols for use of humansubjects were conducted in accordance with the Idaho NationalEngineering and Environmental Laboratory Institutional Review Board.Blood samples were collected in tubes containing the anticoagulant EDTAand were used immediately. Saliva was collected using the SALIVASAMPLER™ saliva collection system. Paired blood and saliva samples wereanalyzed using the blood protocol of Unger and Strauss, supra, and thesaliva AbP test described above.

Four additional volunteers participated in a saliva adulteration studyto assess the effects of various foods and beverages on the AbP assay.The volunteers were given butterscotch and lemon hard candy, sugar andsugar-free gum, sugar and sugar-free cola, and milk chocolate. Aftereating the above, they were asked to collect saliva samples using theprovided saliva samplers. Volunteers were also asked to consume alcohol,drink coffee, eat a food of their choice, and brush their teeth prior togiving samples. A volunteer who was a smoker provided a sample aftersmoking a cigarette. Baseline samples were also collected from thevolunteers.

Monoclonal antibodies against methamphetamine and benzoylecgonine wereconjugated to alkaline phosphatase using the Pierce EZ-LINK™ maleimideactivated alkaline phosphatase kit according to the manufacturer'sprotocols. Unconjugated antibody was separated from the antibody-enzymeconjugate using the FREEZYME™ conjugate purification kit according tothe manufacturer's protocols.

Competitive enzyme linked immunosorbent assays (ELISAs) were developedfor both Cocaine (crystalline tropane alkaloid) and methamphetamine. TheBSA conjugates of methamphetamine or benzoylecgonine were diluted in 50mM carbonate buffer, pH 9.6, and 50 μl was added to each well of a96-well microtiter plate. The plate was incubated overnight at 4° C. toallow the conjugates to bind to the well surfaces. The plate was thenwashed with PBST to remove excess BSA conjugate. Next, 50 μl of eitherCocaine (crystalline tropane alkaloid) or methamphetamine solution inthe concentration range from 0 to 1000 μg/ml was added to the plate and50 μl of either monoclonal anti-benzoylecgonine or anti-methamphetamineconjugated with alkaline phosphatase was added. During this step, theimmobilized BSA drug conjugate competed with the free drug in solutionfor binding sites on the antibodies. After the competition reaction wascomplete, the unbound antibodies and free drug were washed away.Finally, 100 μl of soluble alkaline phosphatase substrate (PNPP)solution was added to the wells to react with the alkaline phosphatasebound to the well surfaces through the anti-drug antibodies. Thereaction was stopped after 20 to 30 minutes by addition of 25 μl of 3 MNaOH, and the absorbance of each well was read at 405 nm using a TecanSpectra microplate reader.

Polyvinylidene fluoride (PVDF) membrane is used in the manufacture ofthe Miragen AbP strips, and was used to assess the feasibility ofbinding the Cocaine (crystalline tropane alkaloid) andmethamphetamine-BSA conjugates to its surface. The PVDF membrane was cutinto strips the same size as those used in the AbP assay. Four stripswere prepared for each drug and 10 μl spots of either drug-BSA conjugatewere placed at three locations on each strip for analysis in triplicate.The strips were dried at 35° C. for one hour prior to use. Non-specificbinding sites on the strips were blocked with PBST containing 1 mg/mLBSA for one hour and then rinsed with PBST. Cocaine (crystalline tropanealkaloid) and methamphetamine solutions were prepared in PBST atconcentrations of 0, 0.1, 10, and 1000 μg/ml. Next, 750 μl of Cocaine(crystalline tropane alkaloid) or methamphetamine solution was added tothe strips and another 750 μl of anti-benzoylecgonine oranti-methamphetamine antibodies conjugated with alkaline phosphatasewere added and allowed to incubate for one hour. During this time acompetitive reaction between the free and the immobilized drug forantibody binding sites took place. The strips were washed to removeunbound antibodies and drugs and the NBT/BCIP substrate was added. Thestrips were allowed to develop for 15 minutes.

A combined AbP-drug assay was prepared by placing 10 μl spots of bothmethamphetamine and benzoylecgonine-BSA conjugate onto the blank bottomportion of the AbP strip and allowing them to dry for one hour at 35° C.Saliva samples from three individuals were collected using ORASURE®samplers. Half of the saliva sample was spiked with 1000 μg/ml ofCocaine (crystalline tropane alkaloid) or methamphetamine. The stripswere blocked with PBST containing 1.0 mg/ml BSA for one hour and rinsedwith PBST. Next, 500 μl of spiked or unspiked saliva was added to thestrips along with alkaline phosphatase conjugated anti-benzoylecgonineand anti-methamphetamine antibodies and allowed to incubate over nightat room temperature. The strips were washed with PBST and the AbP assaywas conducted as described above.

Results and Discussion. The saliva-based AbP assay was optimized throughvariation of reagent concentrations, sample volumes, and incubationtimes. Illustrative results of antibody profiles obtained from salivasamples are shown in FIG. 1. Compared to the blood-based AbP assay, thesaliva assay takes much longer (18 hours versus two hours) and requiresa ten-fold larger amount of sample. This is due to the 100-fold lowerlevels of total antibody present in saliva as compared to blood. Parry,“Tests for HIV and Hepatitis Viruses,” 694 Annals. N.Y. Acad. Sci. 221(1993).

The stability of antibodies present in the saliva samples collectedusing the SALIVA SAMPLER™ or the SALIVETTE™ systems was determined bystorage at −20° C., 4° C., and 25° C. and AbP testing of samples dailyover the period of one week to see if there were any changes in thepatterns observed. Fresh saliva samples from either sampler gave thebest results. The stability over time of samples collected with theSALIVA SAMPLER™ system was superior to samples collected with theSALIVETTE™ system at all temperatures. The preservative storage bufferprovided with the SALIVA SAMPLER™ system appears to prevent antibodydegradation due to bacterial contamination, while the SALIVETTE™ samplerincludes no preservative.

The samples collected with the SALIVA SAMPLER™ system and maintained atroom temperature showed no change in pattern over a five-day period.This result is in contrast to the results obtained with samples storedin a refrigerator, which showed marked deterioration even after a fewhours of storage. It is not clear why this occurred. Frozen samples alsoshowed some deterioration due to damage caused by freeze-thaw cycles,but prolonged storage at freezing temperatures resulted in no furtherdegradation. Since SALIVA SAMPLER™ saliva collection systems hadsuperior storage properties and were easier to use, they were used forthe adulteration studies.

Blood AbP patterns were compared to saliva AbP patterns to determine ifthe ISAs present in those samples were the same. The results showed thatthe patterns obtained from the two different samples differed markedly(FIG. 2). This result was somewhat surprising since saliva is a filtrateof blood, and it was expected that the ISAs present in saliva would bethe same as those present in blood. The different patterns probablyresulted from the isotype of antibody examined in each case. In bloodIgG antibodies were analyzed since they are the most prevalent. Insaliva, IgA antibodies are more prevalent and were analyzed. After theabove result was obtained, saliva samples were also analyzed for IgGantibodies to determine if those patterns would be the same as thosefrom the blood patterns. However, this was unsuccessful due to theextremely low levels of IgG antibodies present in saliva.

The saliva adulteration studies showed that virtually no changesoccurred in the antibody profiles when any of the adulterants werepresent (FIG. 3). In some cases a band might be darker or lighter, butthere appeared to be no missing or additional bands present. Since thiswas a preliminary study, the adulterants examined were easily obtainableitems that might be used during the course of ordinary life. However, asa quick search of the Internet reveals, there are many proposed methodsto beat urine-based drug tests including ingestion of substances and/oradulteration of samples with various substances that are being sold bythese sites. The adulteration results shown here are promising since itappears that the AbP test is not affected by foods that may be commonlyconsumed before taking a saliva test.

Immunoassay tests for both Cocaine (crystalline tropane alkaloid) andmethamphetamine were developed using a direct competitive assay. Ananti-benzoylecgonine antibody was used for the Cocaine (crystallinetropane alkaloid) assay; however, this antibody gave the same responseto Cocaine (crystalline tropane alkaloid) as to benzoylecgonine (theprimary metabolite of Cocaine (crystalline tropane alkaloid)) so it didnot affect the results of the assay. In this assay, a drug present in asample competes for binding sites on enzyme labeled antibodies with aBSA-conjugated drug immobilized to the surface of a well of a microtiterplate. In samples with large drug concentrations, most of theantibody-enzyme conjugate will bind to the drug in solution and will bewashed away during the final step. Therefore, there will be very littleenzyme present in the microtiter plate and the amount of colordevelopment will be low. Conversely, if there is no drug in the sample,the antibodies will bind to the immobilized drugs and stay in the wellsafter the wash step, resulting in strong color development. This resultsin a signal that is inversely proportional to the drug concentration(FIGS. 4 and 5). The linear range for Cocaine (crystalline tropanealkaloid) detection was from 0.1 to 5 μg/ml and for methamphetamine wasfrom 0.1 to 10 μg/ml. This range covers the cutoff values for thesedrugs (0.3 and 1.0 μg/ml, respectively) currently set by the SubstanceAbuse and Mental Health Services Administration. M. Peat and A. E.Davis, Drug Abuse Handbook (CRC Press, Boca Raton, Fla. 1998).

Using the optimum concentrations of BSA-drug conjugates determinedduring the ELISA studies, the drug assays were conducted on the PVDFmembranes. Because of the inverse relationship of the immunoassay todrug concentration, a dark spot was observed when the concentration ofdrugs was low, and spots gradually disappeared as the drug concentrationincreased (FIGS. 6 and 7).

Since the drug test on the PVDF membranes were promising, thefeasibility of combining the two drug tests with the AbP assay wasassessed. Antibody profile patterns from the three individuals did notchange regardless of whether the drug was present or not (FIG. 8). Thisresult shows that the presence of the drugs did not interfere with thereagents used to perform the antibody profiling assay.

EXAMPLE 2

In this example, the procedure of Example 1 is followed except thatfractionated HeLa cell antigens are immobilized on a PVDF membrane in apredetermined pattern as a two-dimensional array. Additionally, Cocaine(crystalline tropane alkaloid) and methamphetamine are immobilized onthe membrane as additional spots on the array. After development ofcolor as described, results are substantially similar to those ofExample 1.

EXAMPLE 3

In this example, the procedure of Example 2 is followed except that thearray is immobilized on a glass slide.

EXAMPLE 4

Assay strips were prepared as in Example 1 and pre-blocked in PBS. Thestrips were then exposed to various amounts of serum ranging from 50 μlto 0.1 μl for 20 minutes. The strips were then placed into a bleachsolution (0.5% v/v sodium hypochlorite) before washing four times withPBS.

In the case of strips undergoing a two-stage antibody layering process,the strips were exposed to a rabbit anti-human IgG for 12 minutes beforebeing washed four times with PBS. These strips were then exposed to agoat anti-rabbit IgG conjugated to alkaline phosphatase for 12 minutes.The strips were then washed and the color developed as outlined inExample 1. Results of two antibody layers can be seen in FIG. 9 whereina readable pattern with some bands missing is visible down to 1microliter of serum and a complete pattern is visible at 3 microlitersof serum.

In the case of strips undergoing a three-stage antibody layeringprocess, the strips were exposed to a rabbit anti-human IgG for 12minutes before being washed four times with PBS. These strips were thenexposed to a goat anti-rabbit IgG for 12 minutes. The strips were thenexposed to a donkey anti-goat IgG conjugated to alkaline phosphatase for12 minutes. The strips were then washed and the color developed asoutlined in Example 1. Results of two antibody layers can be seen inFIG. 10 wherein a readable pattern with some bands missing is visibledown to 0.5 microliter of serum and a complete pattern is visible at 1microliter of serum.

A comparison of FIGS. 9 and 10 shows that a three-stage antibodylayering process has increased sensitivity in providing a readablepattern of individual-specific antibodies over the two-layer process.

EXAMPLE 5

Assay strips were prepared as in Example 1 and pre-blocked in PBS. Thestrips were then exposed to three microliters of serum ranging for 20minutes. The strips were then placed into a bleach solution (0.5% v/vsodium hypochlorite) before washing four times with PBS.

In the case of the strip undergoing a two-stage antibody layeringprocess, the strips were exposed to a rabbit anti-human IgG for 12minutes before being washed four times with PBS. These strips were thenexposed to a goat anti-rabbit IgG conjugated to alkaline phosphatase for12 minutes. The strips were then washed and the color developed asoutlined in Example 1.

In the case of the strips undergoing a three-stage antibody layeringprocess, the strips were exposed to a goat anti-human IgG for 12 minutesbefore being washed four times with PBS. These strips were then exposedto a rabbit anti-goat IgG for 12 minutes. The strips were then exposedto a donkey anti-rabbit IgG conjugated to alkaline phosphatase 12minutes. The strips were then washed and the color developed as outlinedin Example 1.

Results of the two-stage and three-stage antibody layering processes canbe viewed side by side in FIG. 11. Strip A is the three-layer strip andstrip B is the two-layer strip. As can be seen, a much stronger signaland sensitivity were obtained with the three-layer process.

A densitometry study of the two strips was performed and the results arepresented in FIG. 12, where the three-layer strip is the top line andthe two-layer strip is the bottom line. The height of the linesindicates the intensity of the bands. As can be seen in FIG. 12, thethree-layer process has an increased readout for specific bands withouta proportional increase in the background noise. Thus, the three-layerprocess has increased sensitivity over the two-layer process.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

1. A method for analyzing biological material comprisingindividual-specific antibodies, the method comprising: forming an arraycomprising multiple antigens attached to a surface of a solid support ina preselected location pattern; obtaining a sample of a biologicalmaterial having individual-specific antibodies and contacting the arraywith the sample to bind at least a portion of the individual-specificantibodies to the multiple antigens of the array, to form immunecomplexes; washing the array containing the immune complexes; detectingthe immune complexes by the application to the array of at least threeseparate antibodies; and identifying the immune complexes on the array,to obtain an antibody profile.
 2. The method of claim 1, wherein formingan array comprises attaching the multiple antigens to the solid supportthrough a covalent bond.
 3. The method of claim 1, comprising obtaininga sample of a biological material selected from the group of biologicalmaterial consisting of tissue, blood, saliva, urine, perspiration,tears, semen, serum, plasma, amniotic fluid, pleural fluid,cerebrospinal fluid, and combinations thereof.
 4. The method of claim 1,wherein forming the array comprises attaching multiple antigens to asolid support comprising glass or silica.
 5. The method of claim 1,wherein detecting the immune complexes comprises treating the array suchthat the presence of immune complexes at a location is characterized bya color change at the location.
 6. The method of claim 5, whereindetecting the immune complexes comprises obtaining an output using acharge-coupled device and wherein the color change comprisesfluorescence or luminescence emission.
 7. The method of claim 1, whereindetecting the immune complexes further comprises monitoring the arraywith solid state color detection circuitry and comparing color patternsbefore and after detecting the immune complexes.
 8. The method of claim1, wherein detecting the immune complexes further comprises obtaining acolor camera image before contacting the array with the sample and afterdetecting the immune complexes, and analyzing pixel information obtainedfrom the color camera image.
 9. The method of claim 1, wherein detectingthe immune complexes further comprises scanning the array before andafter contacting the array with the sample, wherein the solid support isa surface plasmon resonance chip.
 10. The method of claim 1, whereinforming the array comprises attaching a first subset of antigensconfigured for obtaining an antibody profile and a second subset of atleast one antigen configured for assaying for a selected analyte in thesample.
 11. The method of claim 10, wherein attaching the second subsetof at least one antigen comprises attaching at least one drug.
 12. Themethod of claim 11, wherein attaching at least one drug comprisesattaching a drug selected from the group consisting of marijuana,Cocaine (crystalline tropane alkaloid), methamphetamine, amphetamine,heroin, methyltestosterone, mesterolone and combinations thereof. 13.The method of claim 1, wherein obtaining a sample of a biologicalmaterial comprises obtaining the biological material from a forensicsample.
 14. The method of claim 13, further comprising comparing theantibody profile obtained from the biological material from the forensicsample to an antibody profile prepared from a biological sample obtainedfrom a crime suspect.
 15. A method for analyzing biological materialcomprising individual-specific antibodies, the method comprising:forming an array comprising multiple antigens attached to a surface of asolid support in a preselected location pattern; obtaining a sample of abiological material having individual-specific antibodies and contactingthe array with the sample to bind at least a portion of theindividual-specific antibodies to the multiple antigens of the array, toform immune complexes; washing the array containing the immunecomplexes; contacting the immune complexes with primary antibodiescapable of binding the immune complex, wherein the primary antibodiesare from a different species than the individual-specific antibodies;removing primary antibodies not bound to the immune complexes;contacting the primary antibodies bound to the immune complexes withsecondary antibodies capable of binding the primary antibodies, whereinthe secondary antibodies are from a different species than theindividual-specific antibodies and the primary antibodies; removingunbound secondary antibodies; contacting the secondary antibodies boundto the primary antibodies with tertiary antibodies capable of bindingthe secondary antibodies, wherein the tertiary antibodies are from adifferent species than the individual-specific antibodies, the primaryantibodies, and the secondary antibodies; removing unbound tertiaryantibodies; detecting the primary, secondary, or tertiary antibodies;and identifying the immune complexes on the array, to obtain an antibodyprofile.
 16. A method for detecting a selected drug in a biologicalsample comprising individual-specific antibodies and identifying asource of the biological sample, the method comprising: immobilizingmultiple antigens in a pre-selected pattern on a solid support;immobilizing a detectable amount of a selected drug on the solidsupport, to form an array; providing an antibody-enzyme conjugatecomprising an antibody configured to bind the selected drug and anenzyme that is capable of converting a colorigenic substrate into acolored product; contacting the array with a biological sample, to bindat least some of the multiple antigens with individual-specificantibodies in the biological sample, to form immune complexes;contacting the array with the antibody-enzyme conjugate, wherein theantibody-enzyme conjugate competitively binds to (i) the selected drugimmobilized on the array, to form an immobilized antibody-enzymeconjugate, and (ii) any selected drug that may be present in thebiological sample, to form a soluble drug-antibody-enzyme conjugate;washing the solid support, to remove at least the solubledrug-antibody-enzyme complexes; contacting the solid support with acolorigenic substrate to convert the colorigenic substrate to a coloredproduct using the immobilized antibody-enzyme conjugate; determining anamount of the colored product present, wherein the amount of the coloredproduct may be inversely correlated with an amount of the selected drugin the biological sample; and detecting the immune complexes immobilizedon the solid support by the application to the solid support of at leastthree separate antibodies to form an antibody profile characteristic ofthe source of the biological sample.
 17. The method of claim 16, furthercomprising comparing the antibody profile to one or more candidateantibody profiles from candidate sources, wherein a match of theantibody profile to the one or more candidate antibody profilesidentifies the source of the biological sample.
 18. The method of claim16, wherein immobilizing a detectable amount of the selected drug on thesolid support comprises selecting the selected drug from the groupconsisting of marijuana, Cocaine (crystalline tropane alkaloid),methamphetamine, amphetamine, heroin, methyltestosterone, mesteroloneand combinations thereof.
 19. The method of claim 16, wherein contactingthe array with a biological sample comprises obtaining a biologicalsample from a source selected from the group consisting of tissue,blood, saliva, urine, perspiration, tears, semen, serum, plasma,amniotic fluid, pleural fluid, cerebrospinal fluid, and combinationsthereof.
 20. The method of claim 16, comprising obtaining the biologicalsample from saliva.
 21. The method of claim 16, comprising immobilizingmultiple antigens from a HeLa cell.
 22. The method of claim 16,comprising immobilizing multiple antigens from a random peptide library.23. The method of claim 16, comprising immobilizing multiple antigensfrom an epitope library.
 24. The method of claim 16, comprisingimmobilizing multiple antigens from a random cDNA expression library.25. The method of claim 16, comprising immobilizing multiple antigens onthe solid support, wherein the solid support comprises at least onesubstance selected from the group of substances consisting of glass,silicon, silica, polymeric material, poly(tetrafluoroethylene),poly(vinylidene difluoride), polystyrene, polycarbonate,polymethacrylate, ceramic material, and hydrophilic inorganic material.26. The method of claim 16, comprising immobilizing multiple antigens onthe solid support, wherein the solid support comprises a hydrophilicinorganic material selected from the group consisting of at least one ofalumina, zirconia, titania, nickel oxide.
 27. The method of claim 16,wherein providing the antibody-enzyme conjugate comprises the antibodyconjugated to alkaline phosphatase.
 28. The method of claim 16, whereinproviding the antibody-enzyme conjugate comprises providing the antibodyconjugated to horseradish peroxidase.
 29. The method of claim 16,wherein detecting the immune complexes immobilized on the solid supportby the application to the solid support of at least three separateantibodies comprises: contacting the immune complexes with primaryantibodies capable of binding the immune complex, wherein the primaryantibodies are from a different species than the individual-specificantibodies; removing primary antibodies not bound to the immunecomplexes; contacting the primary antibodies bound to the immunecomplexes with secondary antibodies capable of binding the primaryantibodies, wherein the secondary antibodies are from a differentspecies than the individual-specific antibodies and the primaryantibodies; removing unbound secondary antibodies; contacting thesecondary antibodies bound to the primary antibodies withenzyme-conjugated tertiary antibodies capable of binding the secondaryantibodies, wherein the enzyme-conjugated tertiary antibodies are from adifferent species than the individual-specific antibodies, the primaryantibodies, and the secondary antibodies; removing unboundenzyme-conjugated tertiary antibodies; and detecting boundenzyme-conjugated tertiary antibodies, to detect the immune complexes onthe solid support.
 30. The method according to claim 1, wherein the atleast three separate antibodies comprise: a first antibody capable ofbinding the immune complex; a second antibody capable of binding thefirst antibody; and a third antibody capable of binding the secondantibody.